High-throughput screening (HTS) of small molecule libraries is a widely recognized and increasingly important method for identifying compounds that modulate biological processes. Small molecule compounds thus identified can be powerful molecular probes for dissecting complex biological pathways, and may also be potential leads for development as therapeutics. RNA interference (RNAi) is another powerful tool for probing gene function and, using HTS technologies, can be implemented systematically to screen entire genomes for specific molecular functions or potential therapeutic targets. The ICCB-Longwood Screening Facility (ICCB-L) Research Core provides small molecule and RNAi HTS resources for Harvard Medical School (HMS), HMS-affiliated hospitals, members of the broader Boston-area research community, and other labs nationwide. Here we request funding to purchase a Molecular Devices IsoCyte laser scanning cytometer for use in high-throughput screening at ICCB-L. Although this type of instrument is in use in academic screening centers at other sites in the U.S., no similar instrument currently exists at HMS or in the adjacent research hospitals. As described in this application, the IsoCyte will bring new and much- needed assay capabilities to a large NIH-funded community. The IsoCyte generates low-resolution images of microplate wells. Fluorescence emissions from laser-excited samples are collected using an optical head with a large depth of focus and multiple photomultiplier tubes. A light-scatter mode facilitates generation of images that resemble brightfield images from non-fluorescent samples. Image analysis algorithms run concurrently as images are captured to detect objects (cells) and quantify intensities in up to 4 emission channels. The IsoCyte occupies a useful niche for assay readout between multimode plate readers and screening microscopes. Key features of the IsoCyte relative to plate readers include the greatly enhanced sensitivity of the IsoCyte and the ability to collect single-cell data. The IsoCyte is 5-10 times faster at capturing and analyzing low resolution images than screening microscopes currently in use. Because of its large depth of field, the IsoCyte is ideal for automating imaging of whole organisms such as zebrafish. Such screens are not feasible using conventional screening microscopes. Reflecting the many potential applications of the IsoCyte, this proposal includes specific projects addressing a wide range of topics in the areas of infectious disease, neurobiology, cancer biology, and cell death. Because of its location in the ICCB-L Research Core, the instrument will be well maintained, well managed, and highly accessible to new users as they are recruited.